Investigating xanthine oxidase toxicity modelsin cultured cerebellar granule neurons

RM Therapeutics. Pharmacology

Al-Gonaiah, Majed A. ; Stone, T.W.

University:University of Glasgow

Department:Institute of Neuroscience and Psychology

关键词: Xanthine Oxidase, Xanthine, NADH, superoxide, hydrogen peroxide, hydroxyl radical, cerebellar granule neurons;   

Others  :  http://theses.gla.ac.uk/1057/1/2009AL-Gonaiahphd.pdf

来源: University of Glasgow

【 摘 要 】

In the last few decades, evidence has been accumulating for a role for xanthineoxidoreductase (XOR)-generated toxic reactive oxygen species (ROS) in a variety ofpathological conditions that affect different organ systems. This enzyme in mammalsexists in two inter-convertible forms: xanthine dehydrogenase (XDH) (the predominantintracellular form under physiological conditions) and xanthine oxidase (XO). Acombination of XO and its oxidizable substrate xanthine (X) (or hypoxanthine (HX)) iswidely used as a model to produce ROS and to study their effects in a variety of cellculture studies. However, the effect of the combination of XOR and the reducednicotinamide adenine dinucleotide (NADH) in cell cultures is much less studied. NADHis another oxidizable substrate for XOR that binds to a different site on the enzyme fromthat of X binding.The aim of this project was to investigate some aspects of the in vitro toxicity of XOR,which might provide more insights into its in vivo toxicity. The main investigation wasa comparison between the well studied X / XO and the much less studied NADH / XOtoxicity models. Also, secondary studies were undertaken to investigate those aspects ofX / XO toxicity where there are uncertainties about them.These studies were performed using primary cell cultures. Cell cultures are now widelyused to study different diseases, and although they have their drawbacks, they have theiradvantages over the in vivo studies. For this project, primary cultures of cerebellargranule neurons (CGNs) were used. In the beginning, some problems were encounteredwith CGNs. The main problem was the immediate damage induced to the neurons(including those in the control groups) at the intervention/experiments day (i.e. day 8 or9 after plating) by manipulating the cultures (i.e. aspirating the culture medium, addingtreatment and control vehicles, and adding the restoration medium).After several months of investigation, it was serendipitously discovered that theimmediate damage seen in the neurons (including those in the control groups) whenthey are manipulated at the experiments/intervention day was due to glutamateexcitotoxicity (through activating its N-methyl-D-aspartate (NMDA) receptors). Thesource of glutamate was the fresh serum which is present at 10% V/V in the freshculture medium that is added to the cultures at that day. After solving this problem, itwas possible to conduct reliable experiments to investigate XO toxicity models.Regarding investigating XO toxicity, it was found that both of the X / XO and NADH /XO combinations were toxic to cultures of CGNs. However, the concentration ofNADH needed to cause the toxicity was much higher than that of the other substrate, X,which is in agreement with previous cell-free experiments that showed that NADH is amuch weaker substrate than X for the bovine milk XO used here. Blocking the site of Xbinding on XO prevented X / XO toxicity, but did not prevent NADH / XO toxicity. Onthe other hand, blocking the site of NADH binding prevented both X / XO and NADH/XO toxicities. Another difference between the two systems was that deactivating eithersuperoxide or hydrogen peroxide (both are ROS) generated by XO prevented NADH /XO toxicity, whereas although deactivating hydrogen peroxide prevented X / XOtoxicity, deactivating superoxide generated from this combination did not. In the NADH/ XO system, an extracellular metal contaminant (likely contaminating XOpowder/preparation) seemed to be involved in the toxicity. The two toxicity modelswere similar in the mediation of toxicity by intracellular iron ion. In X / XO toxicity,although superoxide generated extracellularly from the combination has no role in thetoxicity, intracellularly produced superoxide seemed to play a role.Conclusions:1. Culturing/experimental conditions have been optimised for viability studies inCGNs cultures.2. The combination of NADH and XO induces damage to CGNs, where althoughblocking the NADH binding site prevents this damage, blocking the X bindingsite does not. It is feasible that the oxidation of NADH by some forms of XOR(other than the one used here) that are known to be very efficient in oxidizingNADH might produce in vivo toxicity.3. A possibility raised by this study is that a metal (like the metal contaminantproposed to play a role in NADH / XO toxicity in this study) might contribute toXOR toxicity in vivo.4. Intracellular superoxide often mediates XOR toxicity.5. The results add support to many previous studies which suggested thatintracellular hydroxyl radical (or a similar species) is involved in XOR toxicity.